Choose your preferred view mode

Please select whether you prefer to view the MDPI pages with a view tailored for mobile displays or to view the MDPI
pages in the normal scrollable desktop version. This selection will be stored into your cookies and used automatically
in next visits. You can also change the view style at any point from the main header when using the pages with your
mobile device.

Abstract

:
Four new endiandric acid analogues, tsangibeilin C (1), tsangibeilin D (2), tricyclotsangibeilin (3) and endiandric acid M (4), one new lignan, beilschminol B (5) and two new sesquiterpenes, (+)-5-hydroxybarbatenal (6) and (4R,5R)-4,5-dihydroxycaryophyll-8(13)-ene (7), together with four known compounds (8–11), were isolated from the roots of Beilschmiedia tsangii (Lauraceae). The structures of 1–7 were determined by spectroscopic techniques. Among the isolates, endiandric acid M (4) exhibited moderate iNOS inhibitory activity, with an IC50 value of 31.70 μM.

1. Introduction

Over 40 species of Formosan lauraceous plants have been screened for anti-inflammatory activity using an inducible nitric oxide synthase (iNOS) assay. A methanolic extract of the roots of B. tsangii Merr. (Lauraceae) has shown potent inhibition of NO production, with no cytotoxicity against RAW 264.7 cells. B. tsangii is a medium-sized evergreen tree, distributed throughout Tonkin, Vietnam, southern and western China and southern Taiwan. About 200 Beilschmiedia species are found in these tropical regions, including two species in Taiwan [1]. Our previous study reported two new tetrahydrofuran-type lignans, beilschmin A and beilschmin B, two new 1-phenylbutyl benzoates, tsangin A and tsangin B, together with thirteen known compounds isolated from the stem of this plant [2]. One year later, three new epoxyfuranoid lignans, 4α,5α-epoxybeilschmin A, 4α,5α-epoxybeilschmin B and beilschmin D, together with nine known compounds, were obtained from the leaves [3]. More recently, six new endiandric acid analogues, tsangibeilin A, tsangibeilin B, endiandramide A, endiandric acid K, endiandric acid L and endiandramide B, two new lignans, beilschminol A and tsangin C, and six known compounds have been obtained from the roots of this species [4]. In this continuation of our research, four new endiandric acid analogues, tsangibeilin C (1), tsangibeilin D (2), tricyclotsangibeilin (3) and endiandric acid M (4), one new lignan, beilschminol B (5) and two new sesquiterpenes: (+)-5-hydroxybarbatenal (6) and (4R,5R)-4,5-dihydroxycaryophyll-8(13)-ene (7), together with four known compounds (8–11), have been isolated from the roots of this plant. The structures of 1–7 were determined by spectroscopic techniques. This paper elucidates the structures of 1–7 (Figure 1). Endiandric acid M (4) exhibited moderate iNOS inhibitory activity, with an IC50 value of 31.70 μM.

2. Results and Discussion

2.1. Structure Elucidation

Compound 1 was isolated as a light yellowish oil. Its molecular formula was established as C20H26O3 by ESIMS and HRESIMS, with eight unsaturated degrees. The IR spectrum showed absorptions at 1696 cm−1 for carbonyl groups (δ 201.6, C-4 and δ 168.3, C-14) and at 3432 cm−1 for a hydroxy group of carboxylic acid. These findings were supported by 13C NMR spectrum. The 1H, 13C NMR (Table 1), COSY (Figure 2), HSQC and HMBC (Figure 2) spectra of 1 were similar to those of beilschmiedic acid D [5] and also contained 13 skeletal signals of an endiandric acid moiety. The characteristic two cis olefinic protons at δ 5.56 (ddd, J = 10.2, 3.0, 1.8 Hz, H-8) and 5.85 (ddd, J = 10.2, 4.2, 3.0 Hz, H-9) in 1 were similar to those of beilschmiedic acid D, but the signal for another olefinic proton in 1 was shifted upfield to δ 6.70 (d, J = 1.2 Hz, H-5), because a carbonyl group (δC 201.6, C-4) in 1 replaced a methylene group [δ 2.06 (m, Ha-4) and 2.54 (dt, J = 8.8, 3.1 Hz, Hb-4)] in beilschmiedic acid D. The length of the alkyl side chain at C-11 of 1 was two methylenes less than beilschmiedic acid D, as supported by the molecular formula of 1 (C20H26O3). The rigid tetracyclic skeleton was indicated by HMBC correlations, including: H-5 to C-3, C-6, C-7 and C-14, H-3 to C-4, and C-7, H-13 to C-8 and C-10, H-8 to C-6 and C-10, H-9 to C-7, H-2 to C-3, C-4, C-11 and C-13, H-1 to C-3 and C-13, H-12 to C-3, C-9 and C-11 and H-11 to C-9. The relative configuration of 1, rel-(1S,3R,7R,10R,11S,12S,13S), was supported on the basis of NOESY (Figure 3) and structural similarity with beilschmiedic acid D [5]. There are seven chiral centers in 1. However, in view of the observed optical rotation ([α]23D ± 0), 1 should be racemic, as are many endiandric acid analogues [6–9]. According to the above data, the structure of 1 was elucidated and named tsangibeilin C.

Analysis of the ESIMS and HREIMS of 2 revealed a molecular formula of C19H26O3, representing seven degrees of unsaturation, one carbon and one degree of unsaturation less than compound 1 (C20H26O3). The IR spectrum indicated the presence of a hydroxy group (3397 cm−1) and a carbonyl group (1692 cm−1). The rigid tetracyclic skeleton of 2 was the same as that of 1, according to its 1H and 13C NMR spectra (Table 1), inclusive of COSY (Figure 2), NOESY (Figure 3), HSQC and HMBC (Figure 2) experiments. The major differences between 1 and 2 were two hydroxy groups at C-6 and C-7 in 2 were substituted for the carboxyl group (δ 168.3, C-14) and H-7 [δH 3.51 (1H, br s)] at C-6 and C-7 in 1, as supported by HRESIMS, IR and DEPT spectra. The NOESY spectrum (Figure 3) showed correlations between Ha-2, H-3 and H-11, but these three protons showed no correlations with H-1, Hb-2, H-10, H-12 and H-13. This suggested that Ha-2, H-3 and H-11 are on the same side of the molecule, and that H-1, Hb-2, H-10, H-12 and H-13 are on the opposite side of the molecule. The β-orientation of the hydroxy group at C-7 was attributed according to the structural similarity with endiandric acid analogues and biogenetic consideration, where the rings A/B, B/C, C/D and B/D were cis-fusion and ring A/C was trans-fusion [4,8,9]. The relative configuration of 2, rel-(1S,3R,7R,10R,11S,12S,13S) was supported on the basis of NOESY and structural similarity with beilschmiedic acid D [5]. On the basis of the observations above, compound 2 was elucidated and named tsangibeilin D.

Compound 3 was obtained as an optically active light yellowish oil with [α]24D + 36.3 (c 0.024, CHCl3). IR absorption bands at 3422 cm−1 (OH) and 1729 cm−1 (ester carbonyl) were observed. The ESIMS analysis of 3 showed the [M+Na]+ ion at m/z 341, in agreement with the molecular formula of C20H30O3, with six degrees of unsaturation as confirmed by HRESIMS. The 13C NMR (Table 2) and DEPT spectra indicated that 3 contains one methyl, eight methylenes, ten methines and one quaternary carbon. The HSQC and COSY (Figure 2) spectra revealed three fragments, C1-C2-C3-C4-C5-C6, C1-C13-C11-C12-C1 and C-13-C14-C15-C9-C10, and the HMBC (Figure 2) correlations, H-10 to C-9, C-12, C-13 and C-15 and H-9 to C-11, connected the fragments C1-C13-C11-C12-C1 and C-13-C14-C15-C9-C10 to form a cyclohexane ring fused with a cyclobutane ring. The carboxyl group (δ 173.5, C-7) connected the fragment C1-C2-C3-C4-C5-C6 with the cyclohexane ring, as supported by HMBC correlations, H-6 to C-4, C-5 and C-7, H-5 to C-3 and C-7 and H-9 to C-7. According to the above evidence, the carboxyl group, the fragment C1-C2-C3-C4-C5-C6, the cyclohexane ring and the cyclobutane ring become a cyclododecane ring, which is also supported by six degrees of unsaturation. The HMBC spectrum showed correlations between H-1′ and C-1, C-11, C-2′ and C-3′, thus establishing the presence of an alkyl chain (C-1′~C-6′) to be connected at C-12. Finally, the HRESIMS, IR, and DEPT spectra indicated the presence of a hydroxy group located at C-10. The NOESY spectrum (Figure 3) showed correlations between H-9, H-10 and H-12, suggesting these three protons were on the same side of the molecule, but H-1, H-11 and H-13 showed no correlations with H-9, H-10 and H-12, which should therefore be on the opposite side of the molecule. The relative configuration of 3, rel-(1S,9R,10S,11R,12S,13S), was supported on the basis of NOESY and its structural similarity with know endiandric acid analogues. Based on further spectrographic evidence, the structure of 3 was elucidated and designated as tricyclotsangibeilin.

ESIMS and HRESIMS data of compound 4 indicated the molecular formula C22H22O4, four methylenes less than endiandric acid L [4] with 12 degrees of unsaturation, which was the same as endiandric acid L. The UV, IR, 1H NMR (Table 2) and 13C NMR (Table 2) spectra were similar to those of endiandric acid L, except that a methylene group [δ 2.78 (1H, dd, J = 15.6, 7.8 Hz, Ha-1′), 2.81 (1H, br d, J = 7.8 Hz, Hb-1′)] in 4 replaced the pentamethylene group in endiandric acid L. The relative configuration of 4 was supported by NOESY spectrum and its structural similarity with endiandric acid L [4]. Thus, compound 4 was named endiandric acid M. Compounds 1, 2 and 4 were racemic, and all structures were determined by 13C NMR, NOESY, COSY, HSQC and HMBC experiments (see Figures 2 and 3).

Compound 5 was obtained as an optically active light yellowish oil, [α]25D –18.6 (c 0.18, MeOH), and its molecular formula was established as C23H30O7 by ESIMS and HRESIMS. UV absorptions at 209, 240 sh, and 270 nm, in conjunction with a bathochromic shift after the addition of alkali, indicated a phenolic benzenoid moiety. An IR absorption band at 3415 cm−1 (OH) was also observed. The 1H NMR signals for a 7,7′-epoxylignan moiety [δ 1.07 (3H, d, J = 6.0 Hz, H-9′), 1.08 (3H, d, J = 6.0 Hz, H-9), 1.78 (1H, m, H-8), 1.78 (1H, m, H-8′), 4.61 (1H, d, J = 8.8 Hz, H-7) and 4.63 (1H, d, J = 9.2 Hz, H-7′)] were similar to those of beilschminol A [4], except that OCH3-3′ [δ 3.88 (3H, s)] and OCH3-4′ [δ 3.83 (3H, s)] in 5 replaced a methylenedioxy group [δ 5.96 (2H, each d, J = 1.4 Hz)] in beilschminol A. Thus, the planar structure of 5 was proposed to be 3-hydroxy-4,5,3′,4′,5′-pentamethoxy-7,7′-epoxylignan. The relative configuration of 5 was based on the NOESY spectrum and its structural similarity with (7R,8R,7′R,8′R)-3-hydroxy-3′,4′-methylenedioxy-4,5,5′-trimethoxy-7,7′-epoxylign [4]. Thus, compound 5 was elucidated as rel-(7R,8R,7′R,8′R)-3-hydroxy-4,5,3′,4′,5′-pentamethoxy-7,7′-epoxylignan and named beilschminol B.

Compound 6 was isolated as an optically active light yellowish oil, [α]25D + 123.8 (c 0.06, CHCl3). The HRESIMS analysis of 6 gave a quasi-molecular ion peak at m/z 257.1516 [M+Na]+ (calcd. for C15H22O2Na, 257.1517), consistent with a molecular formula of C15H22O2 with five degrees of unsaturation. The IR, 1H, and 13C NMR (Table 3) spectra resembled those of (+)-barbatenal [10], except that a hydroxy group and an oxymethine proton [δ 4.09 (1H, d, J = 3.6, H-5)] in C-5 (δc 71.2) of 6 replaced a methylene [δ 1.65 (1H, dd, J = 21.1, 3.3 Hz, Ha-5), 2.04 (1H, dd, J = 20.1, 2.8 Hz, Hb-5)] in C-5 (δc 41.9) of (+)-barbatenal, resulting in the downshifting of the olefinic proton (δH 6.60, H-4) in 6 from δH 5.87 (H-4) in (+)-barbatenal. The relative configuration of 6 was determined by NOESY spectrum, where Ha-1 showed correlations with H-2, H-12, H-13 and H-14, but no correlation with H-5. It has been suggested that Ha-1, H-2, H-12, H-13 and H-14 are on the same side of the molecule and that Hb-1 and H-5 are on the opposite side of the molecule. Furthermore, H-5 showed a correlation with H-14 due to a spatial distance between these two protons of <4 Å, as supported by simulating the ChemDraw 3D Ultra (version 10.0). Based on the NOESY spectrum, the optical rotation and comparison to values in the literature, the relative configuration of 6 was determined to be rel-(2β, 5α, 12β, 13β, 14β). Hence, the structure of 6 was elucidated and designated as (+)-5-hydroxybarbatenal, which was further confirmed by DEPT, HSQC, NOESY (Figure 3) and HMBC (Figure 2) techniques.

Compound 7 was isolated as an optically active light yellowish oil with [α]25D –23.9 (c 0.032, CHCl3). IR absorption bands at 3396 cm−1 (OH) and 1637 cm−1 (C=C) were observed. From the HRESIMS data, the molecular formula was determined to be C15H26O2 (m/z 261.1828 [M+Na]+; calcd. 261.1830). The 13C NMR (Table 3) and DEPT spectra indicated that 7 contains three methyls, six methylenes, three methines and three quaternary carbons, suggesting a sesquiterpene skeleton. The HSQC and COSY (Figure 2) spectra revealed the correlations from C1-C9, C2-C3 and C-5-C6-C7 and the HMBC (Figure 2) correlations, H-1 to C-3, C-8 and C-11, H-10 to C-8, C-9 and C-11, H-9 to C-1 and C-8, H-7 to C-5, C-6, C-8 and C-9, H-6 to C-4, C-5 and C-8, H-3 to C-4 and C-5 and H-2 to C-1, C-3, C-4 and C-9, connected the fragments C1-C9, C2-C3 and C-5-C6-C7 to form a cyclononane ring and a cyclobutane ring. HMBC correlations, H-15 to C-1, C-10 and C-11, H-14 to C-10, C-11 and C-15, H-12 to C-3, C-4 and C-5, H-13 to C-7, C-8 and C-9, confirmed that three methyl groups were connected at C-4 and C-11 and that a terminal double bond was present at C-8. Finally, the HRESIMS, IR, DEPT spectra indicated two hydroxy groups located at C-4 and C-5, respectively. The NOESY spectrum (Figure 3) showed correlations between Hb-7, H-9, H-12 and H-14, suggesting these three protons were on the same side of the molecule, but H-1, H-5, Ha-7 and H-15 showed no correlations with Hb-7, H-9, H-12 and H-14 and, therefore, should be on the opposite side of the molecule. The structure of 7 was elucidated as (4R, 5R)-4,5-dihydroxycaryophyll-8(13)-ene and identified with its synthetic compound [11]. Though compound 7 has previously been reported as a reduction intermediate [11], our study is the first time to have isolated this compound from a natural source.

The known compounds, 3,4,5-trimethoxybenzaldehyde (8) [12], octahydro-4-hydroxy-3α-methyl-7-methylene-α-(1-methylethyl)-1H-indene-1-methanol (9) [13,14], eudesm-4(15)-ene-1β,6α-diol (10) [15,16] and ursolic acid (11) [17] were identified by comparison of their physical and spectroscopic data with values reported in the literature.

2.2. Anti-inflammatory Activities

Emax (%) and IC50 (μM) values of iNOS inhibitory activity of five compounds, including compounds 4, 5 and 11, and the two compounds, 6β-hydroxystigmast-4-en-3-one and rel-(7S,8S,7′R,8′R)-3,3′,4,4′,5,5′-hexmethoxylignan, reported by Huang et al., 2011 [4], were obtained at the concentration range of 0.1 to 100 μM. Results are shown in Table 4. The technique for the anti-iNOS activity assay was the same as in our previous study [4]. Among the isolates, endiandric acid M (4) exhibited moderate iNOS inhibitory activity, with IC50 value of 31.70 μM. Endiandramide B [4], endiandric acids K [4], L [4] and M, with the same skeleton but with different substituents at C-8, exhibited ascending degrees of iNOS inhibitory activity in this order: endiandramide B (16.40 μM) > endiandric acid M (4) (31.70 μM) > endiandric acid L (39.56 μM) > endiandric acid K (58.21 μM). This suggests that the potency of the substituent at C-8 was an N-isobutylamido group > an α,β-unsaturated carboxylic acid group > a carboxylic acid group. Endiandric acid M (4), with four fewer methylenes than endiandric acid L, showed stronger potency than endiandric acid L. This suggests that fewer methylenes in the alkyl side of endiandric acid analogues result in greater potency of iNOS inhibitory activity.

3.2. Plant Material

Roots of B. tsangii were collected at Mudan, Pingtung County, Taiwan, in April 2009 and identified by one of the authors (I.-S.C.). A voucher specimen (Chen 6120) was deposited in the Herbarium of the School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China.

4. Conclusions

There are about 200 Beilschmiedia species in the tropical regions mentioned earlier, including two species in Taiwan [1]. Except for the two species in Taiwan, previous studies of nine Beilschmiedia species in the world have revealed endiandric acids [5,18,19], flavonoids [20,21], alkaloids [22–24], benzopyrans [18], arylpropanoids [25] and sesquiterpenoids [18]. Several isolates have shown antibacterial [19,21] and antimalarial [22] activities. Phytochemical studies of the two Formosan Beilschmiedia species have been carried out by our group, analyzing the stems [2], the leaves [3] and the roots [4] of B. tsangii, together with the roots [8,9] of B. erythrophloia. Both species possess endiandric acids, 7,7′-epoxylignans, 7′,8′-seco-7,7′-epoxylignans, benzopyrans, arylpropanoids, amides, sesquiterpenoids and steroids. In this study, endiandric acids, 7,7′-epoxylignans, benzenoid, sesquiterpenoids and triterpenoid were also reported. The constituents of the roots of the two Formosan Beilschmiedia species showed 19 new endiandric acid analogues as key secondary metabolites. The roots of other Beilschmiedia species found in other regions have not yet been studied. However, several isolates from two Formosan Beilschmiedia species have shown cytotoxic [2], anti-tubercular [3,9] or anti-inflammatory activities [4] in previous studies. These three bioactivities have not yet been examined in other members of the Beilschmiedia genus.

Acknowledgments

This research was kindly supported by the National Science Council of the Republic of China (NSC 99-2320-B-037-009) and the Kaohsiung Medical University Research Foundation (KMUER013).